Delivery of therapeutic constructs to the posterior eye is an ongoing problem in treating blinding diseases of the retina and retinal pigmented epithelium (RPE). To meet this challenge, our long- term goal is to gain insights into extra- and intracellular trafficking in gene delivery to the RPE. In the past grant period, we demonstrated high in vivo expression of reporter genes in mouse RPE following ocular electroporation with plasmid DNA. Recently, we developed new nanoparticles that have many functional advantages over predecessors due to their specific sizing, core, and shell design. In our preliminary data, we characterized the pharmacokinetics of our new particles. We showed that they reach the posterior segment and transfect retinal cells efficiently, making them ideal for practical gene therapy. Central hypotheses-Hyaluronan coated nanoparticles (HA-NPs) can deliver cargos that other nonviral approaches cannot: 1. They penetrate through interstitial spaces through avidity and adhesiveness. 2. They penetrate cell membranes efficiently through lipid-raft uptake. 3. With aid of inhibitors, they avoid intracellular and cytosolic foreign-DNA host innate defenses. 4. They can be efficiently transported to the nucleus on microtubules. Our studies will show how the HA-NPs overcome these classic barriers to nonviral gene therapy: We will learn the mechanisms of these four key features. These HA-NPs (and knowledge of their mechanism of delivery, and how to manipulate them for optimized delivery) are likely to be effective in gene therapy where other nonviral gene therapy approaches might not. Impact on Field: This research should improve gene delivery to the point that a patient can self- treat with eye drops. The use of topical eye drops to deliver gene therapy agents to the posterior segment and the RPE is transformative. PUBLIC HEALTH RELEVANCE: The relevance of this research to public health is direct. A large proportion of blindness is caused by problems in the back of the eye, which are difficult or impossible to treat now. Many of these diseases could be cured if we could deliver a treatment or agent with topical eye drops. Fortuitously, we have come upon a novel design of nanoparticles that allows efficient entry and expression of a cargo in target cells with no collateral damage. This project tests hypotheses about how this novel nanoparticle selectively overcomes extracellular and intracellular gatekeepers that normally act as barriers and defenses against viral, fungal, and bacterial attacks. Thus, by conducting this project we should learn if these nanoparticles are appropriate for eye drops-based drug delivery.